Cartilage resorption assays measuring type II collagen fragments

ABSTRACT

Immunoassays for measuring type II collagen (cartilage) resorption in vivo employ either one or two antibodies. A first antibody binds to DEKAGGA (SEQ ID NO:21) but not to GGFDEKAGGAQLG (SEQ ID NO:27), where the K symbols refer to lysine or preferably to cross-linking 3-hydroxypyridinium residues. Measurement of analyte binding to the first antibody in serum provides an indication of unmineralized cartilage resorption in vivo. A second antibody binds to GGFDEKAGGAQLG but not to DEKAGGA. Measurement of analyte binding to the second antibody in serum provides an indication of mineralized cartilage resorption in vivo. An indication of total (unmineralized and mineralized) cartilage resorption in vivo is provided by either measurement of analyte binding to the first antibody in urine, or by measurement of the total analyte binding to the first and second antibodies in serum.

This is a continuation-in-part of provisional applications No.60/142,274, filed Jul. 2, 1999, and No. 60/141,574, filed Jun. 29, 1999,and is a continuation-in-part of patent application Ser. No. 09/335,098,filed Jun. 17, 1999 now U.S. Pat. No. 6,255,056, which is acontinuation-in-part of provisional application No. 60/089,823, filedJun. 19, 1998.

This invention was made with government support under one or more ofgrants AR 37318 and AR 36794 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to assays for detecting cross-linked telopeptideanalytes indicative of type II collagen (cartilage) resorption in vivo,and in particular provides immunoassays for measuring and distinguishingbetween resorption of non-mineralized cartilage and mineralizedcartilage, and for measuring total cartilage resorption in vivo.

BACKGROUND OF THE INVENTION

Reference is made to the applicant's prior U.S. Pat. Nos. 4,973,666,5,140,103, 5,300,434, 5,320,970, 5,817,755, and 5,702,909, which areincorporated by reference herein.

Immunoassays are known for detecting telopeptide analytes indicative ofcollagen resorption in vivo. Examples of such type I collagen assays formeasuring bone resorption include: WO 89/04491 (Eyre); WO 89/12824(Robins); WO 91/08478 (Eyre); EP 0505210 A2 (Risteli & Risteli); WO92/21698 (Eyre); WO 94/03813 (Naser et al.); WO 94/14844 (Baylink); WO95/04282 (Naser et al.); WO 95/08115 (Qvist & Bonde); WO 96/12193 (Bonde& Qvist); EP 0718309 A1 (Naser et al.); and WO 96/36645 (Eyre et al.).

Examples of type II collagen telopeptide assays for measuring cartilageresorption include WO 91/08478 (Eyre); WO 95/08115 (Qvist & Bonde); andWO 96/12193 (Bonde & Qvist).

Examples of type III collagen telopeptide assays include WO 88/08980(Risteli & Risteli) and WO 91/08478 (Eyre).

The following patent disclosures are also of interest: U.S. Pat. No.4,628,027 (Gay) discloses in vitro diagnostic methods using monoclonalantibodies against connective tissue proteins. U.S. Pat. No. 5,316,914(Oshima et al.) discloses an enzyme sandwich immunoassay of human typeIII, IV, and VI collagens applicable to diagnosis of hepatic diseases.WO 94/14070 (Poole & Hollander) discloses an immunoassay for themeasurement of collagen cleavage in cartilage. WO 94/18563 (Barrach etal.) discloses sandwich immunoassay methods for detecting type IIcollagen derived peptides associated with arthritis.

Of particular interest is the applicant's prior internationalpublication, WO 91/0478, which discloses the following urinaryresorption products of type II collagen:

wherein the parentheses indicate optional amino acid residues, and thecross-linking residue depicted as Hyl-Hyl-Hyl is hydroxylysylpyridinoline (HP), a natural 3-hydroxypyridinium residue present inmature collagen fibrils of various tissues. These analytes derive fromthe C-terminal cross-linked telopeptide domain of type II collagen andso are collectively referred to herein as “col2CTx”.

The present disclosure also adopts the one-letter symbol form of aminoacid shorthand; thus, the Glu-Hyl-Gly-Pro-Asp-(Pro)-(Leu) telopeptidecomponents of the analytes shown above are referred to hereinafter asEKGPD (SEQ ID NO:1), EKGPDP (SEQ ID NO:2), and EKGPDPL (SEQ ID NO:3).Furthermore, as used herein the symbol “K” represents either lysine, inthe case of linear peptides, or a cross-linking 3-hydroxypyridiniumresidue selected from among hydroxylysyl pyridinoline (HP) and lysylpyridinoline (LP).

The applicant and colleagues have also described col2CTx in recentabstracts:

Eyre et al., Bone Miner. Res. 11(S1): S413, 1996, describes cross-linkedtelopeptides from collagen types I, II, and III in human urine.

Atley et al., Arth. Rheum. 40(9S):584, 1997, reports that RS-130830, aselective inhibitor of collagenase-3, blocks the release ofhydroxyproline and a metalloproteinase (MMP) specific neoepitope,col2CTx, from bovine cartilage exposed to IL-1α.

Atley et al., Trans. Orthop. Res. Soc., New Orleans, 1998, reportsmatrix metalloproteinase-mediated release of immunoreactive telopeptidesfrom cartilage type II collagen. The aim of this study was to evaluatewhether an immunoassay based on a monoclonal antibody (mAb) 2B4, whichrecognizes a domain of the α1(II) C-telopeptide EKGPDP, measures MMPcleavage products in cartilage. Referring to FIG. 1, mAb 2B4 recognizesthe in vitro product of matrilysin digestion AFAGLGPREKGPDP (SEQ IDNO:4) of synthetic peptide AFAGLGPREKGPDPLQYMRA (SEQ ID NO:5), but notAFAGLGPREKGPDPLQ (SEQ ID NO:6), AFAGLGPREKGPDPLQY (SEQ ID NO:7), LQYMRA(SEQ ID NO:8), or YMRA (SEQ ID NO:9). The authors also noted detectionof mAb 2B4 immunoreactivity in synovial fluid, serum, and urine. Theyconcluded that this 2B4 epitope has the potential to be a useful markerof type II collagen resorption in vivo.

Moskowitz et al., Amer. Coll. Rheum., San Diego, Calif., Nov. 8-12,1998, reports that the type II collagen C-telopeptide 2B4 epitope is amarker for cartilage resorption in familial osteoarthrosis.

Eyre et al., Combined Orthopaedic Research Societies Meeting, Vittel,France, Sept. 28-30, 1998, discusses biochemical markers of bone andcollagen degradation, including a cross-linked telopeptide product oftype II collagen degradation in urine (col2CTx).

Atley et al., Combined Orthopaedic Research Societies Meeting,Hamamatsu, Japan, 1998, discusses collagen type II cross-linkedtelopeptides, a promising marker for cartilage degradation in arthritis.

In addition, monoclonal antibodies to the C-telopeptide of type IIcollagen as diagnostic markers for rheumatoid arthritis are reportedlyunder development at Osteometer Biotech A/S. BioWorld International,page 3, Dec. 3, 1997.

The entire disclosures of the prior scientific and patent publicationscited in this patent application are incorporated by reference herein.

SUMMARY OF THE INVENTION

The invention provides improved assays for measuring type II collagen(cartilage) resorption in vivo. The subject immunoassays are useful fordistinguishing between resorption of non-mineralized cartilage andmineralized cartilage, and for measuring total cartilage resorption invivo. The disclosed immunoassays employ either one or a combination oftwo antibodies. A first antibody binds to DEKAGGA (SEQ ID NO:21) but notto GGFDEKAGGAQLG (SEQ ID NO:27), where the K symbols refer to lysineresidues and/or preferably to cross-linking 3-hydroxypyridiniumresidues. Measurement of analyte binding to the first antibody in serumprovides an indication of non-mineralized cartilage resorption in vivo.

A second antibody binds to GGFDEKAGGAQLG but not to DEKAGGA, where the Ksymbols also preferably refer to cross-linking 3-hydroxypyridiniumresidues. Measurement of analyte binding to the second antibody in serumprovides an indication of mineralized cartilage resorption in vivo.

An indication of total (non-mineralized and mineralized) cartilageresorption in vivo is provided by either measurement of analyte bindingto the first antibody in urine, or by measurement of the total analytebinding to the first and second antibodies in serum.

The subject cartilage markers are preferably measured in conjunctionwith bone collagen resorption markers selected from among amino-terminaltelopeptides of type I collagen, carboxy-terminal telopeptides of type Icollagen, and free lysyl pyridinoline (deoxypyridinoline) cross-links.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

By way of introduction, the monoclonal antibody 2B4 recognizes aC-terminal epitope in the sequence EKGPDP. The antibody requires theC-terminal proline to be free for any binding (i.e., Pro-COOH) andprefers K to be derivatized (e.g., cross-linked) through its side-chain(epsilon) amino group. The presence of extra amino acids C-terminal tothe proline prevents mAb 2B4 binding to this peptide domain.

Various matrix metalloproteinases (MMPs) including collagenases (MMP1and MMP13), gelatinases (MMP2 and 9), stromelysin (MMP3), and matrilysin(MMP7) can generate the mAb 2B4 epitope from cartilage type II collagenor a synthetic C-telopeptide domain (AFAGLGPREKGPDPLQYMRA). Matrilysin(MMP7) is the most potent based on in vitro studies with the recombinantenzymes.

Cathepsin K, however, is unable to cleave the P-L bond required for 2B4epitope release, but generates a longer fragment, EKGPDPLQ (SEQ IDNO:10). This is significant because cathepsin K is a protease expressedessentially only by osteoclasts and is the key enzyme responsible forthe resorption of the calcified collagen when osteoclasts resorb bone.Osteoclasts (or a very similar cell) are also responsible for degradingcalcified cartilage. Calcified cartilage is formed transiently in thegrowth plates of growing animals and is also present in the adultskeleton at the interfaces between bone and cartilage in all types ofjoints. In arthritis, accelerated resorption and bony remodeling willresult in extensive resorption of calcified cartilage by osteoclasts.This process can release into the bloodstream collagen type IIC-telopeptides (EKGPDPLQ) that are not recognized by 2B4.

Since the urinary form of the type II collagen telopeptide analyte isEKGPDP or smaller, proteases in the kidney and/or liver remove theC-terminal -LQ before release into the urine. Therefore, two sources ofimmunoreactive col2CTx are can be defined: (1) direct generation bychondrocytes activated to degrade their matrix collagen by MMPs, and (2)secondary generation in the kidney and/or liver from the circulatingproducts of osteoclastic resorption of calcified cartilage.

In arthritis (rheumatoid arthritis (RA) and osteoarthritis (OA)) MMPsare known to be key agents in the destruction of the collagenousmatrices of joint cartilages. In both forms of arthritis, bone structureis also greatly affected. In RA, osteoporosis is a common consequenceboth directly from the disease process itself through the effects ofinflammatory cytokines and as a side effect of corticosteroid therapy.The accelerated bone resorption will be most pronounced in involvedjoints and will include removal of the calcified cartilage underlyingjoint surfaces. In OA, osteophytes (bony outgrowths) often featureprominently in the joint pathology. Here, too, calcified cartilage willbe formed and then degraded (the latter by osteoclasts) as part ofosteophyte growth. Therefore to monitor and manage both major forms ofarthritis it would be desirable to differentially measure MMP-mediatedand osteoclast-mediated (cathepsin K) collagen type II destruction. Inblood or synovial fluid, measuring col2CTx (EKGPDP) can index the formeractivity, whereas measuring the osteoclast product (EKGPDPLQ) in bloodcan index the latter. In addition, urinary col2CTx can provide an indexof total resorption (both non-mineralized and mineralized cartilage).Such urine assays preferably include the steps of determining thecreatinine content of the urine sample and correlating the ratio of thedetected binding of the antibody to the creatinine content in order toprovide a urinary index of total type II collagen resorption independentof urine volume.

The subject differential measurements of EKGPDP and EKGPDPLQimmunoreactivities have diagnostic value in the assessment of diseaseactivity in the individual patient. In combination with measurement of atype I collagen marker to assess total bone resorption activity, and/ormeasurement of a type III collagen marker to assess other sources ofnon-mineralized connective tissue breakdown, an appropriate therapy canbe prescribed and its desired effect monitored. Similarly, suchdifferential assays can be used to identify new drug candidates (invitro and in animals) and to provide surrogate markers in clinicaltrials for demonstrating desired beneficial effects on target tissuesand cellular processes.

Representative assays for type I collagen resorption markers for thispurpose include amino-terminal telopeptides of type I collagen(Osteomark®, Ostex International, Seattle, Wash.), carboxy-terminaltelopeptides of type I collagen (CrossLaps®-D, Osteometer Biotech,Herlev, Denmark), and free lysyl pyridinoline cross-links (Pyrilinks®,Metra Biosystems, Mountain View, Calif.).

Representative assays for type III collagen resorption markers includeamino-terminal telopeptides of type III collagen and carboxy-terminaltelopeptides of type III collagen. Such assays are disclosed in U.S.Pat. Nos. 5,532,169, 5,641,687, and 6,010,862. Type III collagenresorption markers can be used to assess vascular collagen degradation(atherosclerosis), lung tissue degradation (e.g., emphysema and otherdestructive lung diseases), muscle wastage, frailty syndromes of theelderly, liver disease, hyperthyroidism, secondary effects of diabeteson connective tissue, and other inflammatory and infectious conditionsthat accelerate total bone resorption activity.

The invention also provides antibodies directed against othercross-linked carboxy-telopeptide degradation products of type IIcollagen, as well as linear peptides (SEQ ID NOS 11-20) synthesized tomatch the telopeptide components of the degradation products, and assaysfor cartilage degradation using the antibodies and linear peptides.

SEQ Body Fluid¹ ID Synovial α1(II)C Telopeptide Sequence NO fluid BloodUrine             EKGPD 1 — N N + M              EKGPDP 2 N N N + M             EKGPDPL 3 N N N + M              EKGPDPLQ 10 — M —        LGPREKGPDP 11 N N —         LGPREKGPDPLQ 12 N N —        LGPREKGPDPLQY 13 N N —      FAGLGPREKGPDP 14 N N —     FAGLGPREKGPDPLQ 15 N N —      FAGLGPREKGPDPLQY 16 N N —IDMSAFAGLGPREKGPDP 17 N N — IDMSAFAGLGPREKGPDPLQ 18 N N —IDMSAFAGLGPREKGPDPLQY 19 N N —             EKGPDPLQYMR 20 N N —¹Cartilage Source: N = non-mineralized cartilage; M = mineralizedcartilage; N + M = total cartilage degradation; and — = absent or onlytrace amounts.

The principal col2CTx degradation product is cross-linked EKGPDP. Thefurther degradation product cross-linked EKGPD is usually present inurine in lower amounts. The cross-linked EKGPDPL degradation product isa minor component relative to cross-linked EKGPDP.

Higher than normal levels of cross-linked EKGPDPLQ in blood can indicatemineralized cartilage degradation associated with active osteophyteformation in osteoarthritis.

The telopeptide sequences represented by SEQ ID NOS 11-13 result frommetalloproteinase (MMP) cleavage of the G—L bond at the amino end of theα1(II)C telopeptide (SEQ ID NO 5), in combination with MMP cuts of theP—L, Q—Y, or Y—M bonds at the carboxy end of the telopeptide. SEQ ID NOS14-16 represent related telopeptides from MMP cleavage between the A—Fbond. SEQ ID NOS 17-19 represent longer telopeptides from MMP cleavagebetween the G—I bond. The amino ends of these longer telopeptides aregenerally metabolized further in the lymph nodes, liver, and/or kidney.

The telopeptide sequence represented by SEQ ID NO 20 results from serineprotease cleavage of the R—E bond amino terminal to the cross-linking Kresidue, in combination with serine protease cleavage of the R—A bond atthe carboxy end of the telopeptide.

Such cross-linking degradation products of SEQ ID NOS 11-19 (wherein thetwo telopeptide components are independently selected from among SEQ IDNOS 11-19), and SEQ ID NO 20, generally result from degradation ofnon-mineralized cartilage and are primarily found in synovial fluid andblood, and to a lesser extent in urine. Higher than normal levels ofthese degradation products in body fluids indicate active cartilagedegradation that may correlate with rheumatoid arthritis,osteoarthritis, and other arthritides.

The invention also provides antibodies directed against cross-linkedamino-telopeptide degradation products of type II collagen, as well aslinear peptides (SEQ ID NOS 21-31) synthesized to match the telopeptidecomponents of the degradation products, and assays for cartilagedegradation using the antibodies and linear peptides.

SEQ Body Fluid¹ ID Synovial α1(II)C Telopeptide Sequence¹ NO fluid BloodUrine       DEKAGGA 21 N N N + M       DEKAGGAQ 22 N N N + M      DEKAGGAQL 23 N N N + M      FDEKAGGA 24 N N N + M      FDEKAGGAQ25 N N N + M      FDEKAGGAQL 26 N N N + M    GGFDEKAGGAQLG 27 — M —JMAGGFDEKAGGAQLG 28 — M — JMAGGFDEKAGGAQLGV 29 N N —      FDEKAGGAQLGV30 N N —       DEKAGGAQLGV 31 N N — ¹J = pyroglutamic acid, i.e., whollycyclized pyrrolidone carboxylic acid (5-oxo-2-pyrrolidinecarboxylicacid). ²Cartilage Source: N = non-mineralized cartilage; M = mineralizedcartilage; N + M = total cartilage degradation; and — = absent or onlytrace amounts.

The telopeptide sequences represented by SEQ ID NOS 21-23 result frommetalloproteinase (MMP) cleavage of the F—D bond at the amino end of theα1(II)N telopeptide, in combination with MMP cuts of the A—Q, Q—L, orL—G bonds at the carboxy end of the telopeptide. SEQ ID NOS 24-26represent related telopeptides from MMP cleavage between the G—F bond atthe amino end. The amino and carboxy ends of the longer telopeptides(SEQ ID NOS 22-26) are generally metabolized further in the lymph nodes,liver, and/or kidney. As a result, the principal col2NTx degradationproducts in urine are cross-linked DEKAGGA and FDEKAGGA.

The telopeptide sequences represented by SEQ ID NOS 27-28 result fromcathepsin K cleavage of the G—V bond at the carboxy end of thetelopeptide, in combination with cathepsin K cleavage of the A—G bond atthe amino end of the telopeptide in the case of SEQ ID NO 27. In thecase of SEQ ID NO 28 the amino terminal J residue is blocked. Higherthan normal levels of cross-linked GGFDEKAGGAQLG and QMAGGFDEKAGGAQLG inblood can indicate mineralized cartilage degradation associated withactive osteophyte formation in osteoarthritis. Proteases in the kidneyand/or liver remove the carboxy-terminal G residue before release intothe urine.

The telopeptides represented by SEQ ID NOS 29-31 result frommetalloproteinase cleavage of the V—M bond at the carboxy end of thetelopeptide, in combination with MMP cleavage of the G—F or F—D bond atthe amino end of the telopeptide in the case of SEQ ID NOS 30-31. Largerdegradation products comprising SEQ ID NOS 29-31 plus some additionalhelical sequence(s) may also be present in synovial fluid and blood.These cross-linked telopeptides are generally metabolized further in thelymph nodes, liver, and/or kidney, and consequently occur only in traceamounts in urine.

As indicated above, cross-linked α1(II)N telopeptides represented by SEQID NOS 27-31 are produced at the site(s) of cartilage degradation butdegraded further during passage through the lymph nodes, liver, and/orkidney. The resulting smaller degradation products have two telopeptidecomponents that may be independently selected from among SEQ ID NOS21-26. Higher than normal levels of these degradation products in urineindicate total cartilage degradation that may correlate with rheumatoidarthritis, osteoarthritis, and other arthritides.

Thus the invention provides, in a first embodiment, an improved methodof analyzing a body fluid sample for the presence of an analyteindicative of a physiological condition, including the steps ofcontacting the body fluid sample with an antibody which binds to theanalyte, detecting binding of the antibody in the body fluid sample, andcorrelating any detected binding to the physiological condition, theimprovement comprising contacting a serum sample with an antibody whichbinds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding to resorption of non-mineralized typeII collagen in vivo.

In a second embodiment, the invention provides an improved method ofanalyzing a body fluid sample for the presence of an analyte indicativeof a physiological condition, including the steps of contacting the bodyfluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a serum sample with an antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding to resorption of mineralized type IIcollagen in vivo.

In a third embodiment, the invention provides an improved method ofanalyzing a body fluid sample for the presence of an analyte indicativeof a physiological condition, including the steps of contacting the bodyfluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a urine sample with an antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding to resorption of both non-mineralizedand mineralized type II collagen in vivo.

In a fourth embodiment, the invention provides an improved method ofanalyzing a body fluid sample for the presence of an analyte indicativeof a physiological condition, including the steps of contacting the bodyfluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a serum sample with a first antibody which bindsto

and contacting the serum sample with a second antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating the total detected binding of the first and secondantibodies to resorption of both mineralized and non-mineralized type IIcollagen in vivo.

In a fifth embodiment, the invention provides an improved method ofanalyzing a body fluid sample for the presence of an analyte indicativeof a physiological condition, including the steps of contacting the bodyfluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a serum sample with a first antibody which bindsto

and contacting a urine sample with a second antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding of the first antibody to resorption ofmineralized type II collagen in vivo, and any detected binding of thesecond antibody to resorption of both mineralized and non-mineralizedtype II collagen in vivo.

The term “antibody” in this disclosure is meant to encompass polyclonalantibodies, monoclonal antibodies, and antigen-binding fragmentsthereof. Such EKGPDPLQ-binding and EKGPDP-binding antibodies, orGGFDEKAGGAQLG-binding and DEKAGGA-binding antibodies, can be produced bystandard methods well known in the art. A representative protocolfollows.

EKGPDPLQ hybridoma development: Female RFB/DnJ mice are injectedintraperitoneal (ip) with an emulsion of complete Freund's adjuvant andEKGPDPLQ synthetic peptide cross-linked to the carrier protein keyholelimpet hemacyanin (KLH) via glutaraldehyde (KLH-EKOPDPLQ). One monthlater a booster injection of KLH-EKGPDPLQ is given ip in an emulsionwith incomplete Freund's adjuvant. Approximately eight weeks later themice are sacrificed and splenocytes fused, by polyethylene glycol, tothe FOX-NY murine myeloma cell line (ATCC CRL-1732). Fusion cells aregrown in 96-well plates in selection media containing adenine,aminopterin and thymidine (AAT).

Approximately one week after fusion hybridoma colony culturesupernatants are tested in ELISA for reactivity to the EKGPDPLQsynthetic peptide cross-linked via glutaraldehyde to bovine serumalbumin (BSA-EKGPDPLQ). Hybridoma colonies producing antibody reactivewith BSA-EKGPDPLQ are secondarily tested for specificity of reaction inELISA against other synthetic peptide sequences. Among those tested areEKGPDP synthetic peptide cross-linked to BSA via glutaraldehyde(BSA-EKGPDP). Hybridoma colonies that react positively to BSA-EKGPDPLQand negatively to BSA-EKGPDP are cryopreserved. Representativehybridomas are cloned to monoclonality by the limited dilution method.

In competition ELISA, human serum specimens as well as solubleBSA-EKGPDPLQ are found to compete with solid phase BSA-EKGPDPLQ forcandidate antibodies.

A cathepsin K digest of type II collagen can also be prepared forimmunization and screening purposes. Proteoglycans are extracted from atissue sample of human articular cartilage with a protein denaturant,such as 4 M guanidine HCl. The residue is washed with water and digestedwith recombinant cathepsin K (37° C., pH 5.8). The digest isfractionated by high performance liquid chromatography, for example byreverse phase, and fractions containing pyridinoline cross-links aremonitored by fluorescence emission at 390 nm (297 nm excitation). Thefluorescent fraction containing the cross-linked C-telopeptides ofsequence EKGPDPLQ is identified by N-terminal sequence analysis. Thisfraction can be used for screening purposes and for validation ofantibody specificity.

The subject antibodies can be employed in a variety of immunoassayformats that are well known in the art. For example, see: Harlow & Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, pp.553-612, 1988; Principles and Practice of Immunoassay, Price & Newman(Eds.), Stockton Press, 1991; and Immunoassay, Diamandis & Christopoulos(Eds.), Academic Press, 1996.

While the invention has been described in conjunction with preferredembodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the subject matter set forth herein.Hence, the invention can be practiced in ways other than thosespecifically described herein.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 31 <210> SEQ ID NO 1 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(5) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 1 Glu Lys Gly Pro Asp 1 5 <210> SEQ ID NO 2<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(5) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 2 Glu Lys Gly Pro Asp Pro 1 5 <210> SEQ ID NO 3<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(7) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 3 Glu Lys Gly Pro Asp Pro Leu 1 5 <210> SEQ IDNO 4 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(14) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 4 Ala Phe Ala Gly Leu Gly Pro Arg Glu Lys GlyPro Asp Pro 1 5 10 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE<222> LOCATION: (1)..(20) <223> OTHER INFORMATION: ssyn- corr. toC-terminal telopeptide seq. of hu. type II collagen <400> SEQUENCE: 5Ala Phe Ala Gly Leu Gly Pro Arg Glu Lys Gly Pro Asp Pro Leu Gln 1 5 1015 Tyr Met Arg Ala 20 <210> SEQ ID NO 6 <211> LENGTH: 16 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE<222> LOCATION: (1)..(16) <223> OTHER INFORMATION: syn- corr. toC-terminal telopeptide seq. of hu. type II collagen <400> SEQUENCE: 6Ala Phe Ala Gly Leu Gly Pro Arg Glu Lys Gly Pro Asp Pro Leu Gln 1 5 1015 <210> SEQ ID NO 7 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION:(1)..(17) <223> OTHER INFORMATION: syn- corr. to C-terminal telopeptideseq. of hu. type II collagen <400> SEQUENCE: 7 Ala Phe Ala Gly Leu GlyPro Arg Glu Lys Gly Pro Asp Pro Leu Gln 1 5 10 15 Tyr <210> SEQ ID NO 8<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(6) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 8 Leu Gln Tyr Met Arg Ala 1 5 <210> SEQ ID NO 9<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(4) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 9 Tyr Met Arg Ala 1 <210> SEQ ID NO 10 <211>LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(8) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 10 Glu Lys Gly Pro Asp Pro Leu Gln 1 5 <210>SEQ ID NO 11 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(10)<223> OTHER INFORMATION: syn- corr. to C-terminal telopeptide seq. ofhu. type II collagen <400> SEQUENCE: 11 Leu Gly Pro Arg Glu Lys Gly ProAsp Pro 1 5 10 <210> SEQ ID NO 12 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222>LOCATION: (1)..(12) <223> OTHER INFORMATION: syn- corr. to C-terminaltelopeptide seq. of hu. type II collagen <400> SEQUENCE: 12 Leu Gly ProArg Glu Lys Gly Pro Asp Pro Leu Gln 1 5 10 <210> SEQ ID NO 13 <211>LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(13) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 13 Leu Gly Pro Arg Glu Lys Gly Pro Asp Pro LeuGln Tyr 1 5 10 <210> SEQ ID NO 14 <211> LENGTH: 13 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222>LOCATION: (1)..(13) <223> OTHER INFORMATION: syn- corr. to C-terminaltelopeptide seq. of hu. type II collagen <400> SEQUENCE: 14 Phe Ala GlyLeu Gly Pro Arg Glu Lys Gly Pro Asp Pro 1 5 10 <210> SEQ ID NO 15 <211>LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(15) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 15 Phe Ala Gly Leu Gly Pro Arg Glu Lys Gly ProAsp Pro Leu Gln 1 5 10 15 <210> SEQ ID NO 16 <211> LENGTH: 16 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:PEPTIDE <222> LOCATION: (1)..(16) <223> OTHER INFORMATION: syn- corr. toC-terminal telopeptide seq. of hu. type II collagen <400> SEQUENCE: 16Phe Ala Gly Leu Gly Pro Arg Glu Lys Gly Pro Asp Pro Leu Gln Tyr 1 5 1015 <210> SEQ ID NO 17 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION:(1)..(18) <223> OTHER INFORMATION: syn- corr. to C-terminal telopeptideseq. of hu. type II collagen <400> SEQUENCE: 17 Ile Asp Met Ser Ala PheAla Gly Leu Gly Pro Arg Glu Lys Gly Pro 1 5 10 15 Asp Pro <210> SEQ IDNO 18 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(20) <223>OTHER INFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. typeII collagen <400> SEQUENCE: 18 Ile Asp Met Ser Ala Phe Ala Gly Leu GlyPro Arg Glu Lys Gly Pro 1 5 10 15 Asp Pro Leu Gln 20 <210> SEQ ID NO 19<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(21) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 19 Ile Asp Met Ser Ala Phe Ala Gly Leu Gly ProArg Glu Lys Gly Pro 1 5 10 15 Asp Pro Leu Gln Tyr 20 <210> SEQ ID NO 20<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(11) <223> OTHERINFORMATION: syn- corr. to C-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 20 Glu Lys Gly Pro Asp Pro Leu Gln Tyr Met Arg1 5 10 <210> SEQ ID NO 21 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222>LOCATION: (1)..(7) <223> OTHER INFORMATION: syn- corr. to N-terminaltelopeptide seq. of hu. type II collagen <400> SEQUENCE: 21 Asp Glu LysAla Gly Gly Ala 1 5 <210> SEQ ID NO 22 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE<222> LOCATION: (1)..(8) <223> OTHER INFORMATION: syn- corr. toN-terminal telopeptide seq. of hu. type II collagen <400> SEQUENCE: 22Asp Glu Lys Ala Gly Gly Ala Gln 1 5 <210> SEQ ID NO 23 <211> LENGTH: 9<212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: PEPTIDE <222> LOCATION: (1)..(9) <223> OTHER INFORMATION: syn-corr. to N-terminal telopeptide seq. of hu. type II collagen <400>SEQUENCE: 23 Asp Glu Lys Ala Gly Gly Ala Gln Leu 1 5 <210> SEQ ID NO 24<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(8) <223> OTHERINFORMATION: syn- corr. to N-terminal telopeptide seq. of hu. type IIcollagen <400> SEQUENCE: 24 Phe Asp Glu Lys Ala Gly Gly Ala 1 5 <210>SEQ ID NO 25 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(9)<223> OTHER INFORMATION: syn- corr. to N-terminal telopeptide seq. ofhu. type II collagen <400> SEQUENCE: 25 Phe Asp Glu Lys Ala Gly Gly AlaGln 1 5 <210> SEQ ID NO 26 <211> LENGTH: 10 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222>LOCATION: (1)..(10) <223> OTHER INFORMATION: syn- corr. to N-terminaltelopeptide seq. of hu. type II collagen <400> SEQUENCE: 26 Phe Asp GluLys Ala Gly Gly Ala Gln Leu 1 5 10 <210> SEQ ID NO 27 <211> LENGTH: 13<212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: PEPTIDE <222> LOCATION: (1)..(13) <223> OTHER INFORMATION:syn- corr. to N-terminal telopeptide seq. of hu. type II collagen <400>SEQUENCE: 27 Gly Gly Phe Asp Glu Lys Ala Gly Gly Ala Gln Leu Gly 1 5 10<210> SEQ ID NO 28 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(16)<223> OTHER INFORMATION: syn- corr. to N-terminal telopeptide seq. ofhu. type II collagen <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Xaa is pyroglutamic acid <400> SEQUENCE: 28 XaaMet Ala Gly Gly Phe Asp Glu Lys Ala Gly Gly Ala Gln Leu Gly 1 5 10 15<210> SEQ ID NO 29 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(17)<223> OTHER INFORMATION: syn- corr. to N-terminal telopeptide seq. ofhu. type II collagen <221> NAME/KEY: PEPTIDE <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Xaa is pyroglutamic acid <400> SEQUENCE: 29 XaaMet Ala Gly Gly Phe Asp Glu Lys Ala Gly Gly Ala Gln Leu Gly 1 5 10 15Val <210> SEQ ID NO 30 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: PEPTIDE <222> LOCATION:(1)..(12) <223> OTHER INFORMATION: syn- corr. to N-terminal telopeptideseq. of hu. type II collagen <400> SEQUENCE: 30 Phe Asp Glu Lys Ala GlyGly Ala Gln Leu Gly Val 1 5 10 <210> SEQ ID NO 31 <211> LENGTH: 11 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:PEPTIDE <222> LOCATION: (1)..(11) <223> OTHER INFORMATION: syn- corr. toN-terminal telopeptide seq. of hu. type II collagen <400> SEQUENCE: 31Asp Glu Lys Ala Gly Gly Ala Gln Leu Gly Val 1 5 10

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. In a method of analyzinga body fluid sample for the presence of an analyte indicative of aphysiological condition, comprising the steps of contacting the bodyfluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a serum sample with an antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding to resorption of unmineralized type IIcollagen in vivo.
 2. The method of claim 1, further comprising the stepof contacting the serum sample with a second antibody which binds to abone resorption marker selected from among amino-terminal telopeptidesof type I collagen, carboxy-terminal telopeptides of type I collagen,and free lysyl pyridinoline cross-links, and correlating any detectedbinding of the second antibody in the sample to bone resorption in vivo.3. The method of claim 1, further comprising the step of contacting theserum sample with a second antibody which binds to a type III collagenresorption marker selected from among amino-terminal telopeptides oftype III collagen and carboxy-terminal telopeptides of type IIIcollagen, and correlating any detected binding of the second antibody inthe sample to type III collagen resorption in vivo.
 4. In a method ofanalyzing a body fluid sample for the presence of an analyte indicativeof a physiological condition, comprising the steps of contacting thebody fluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a serum sample with an antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding to resorption of mineralized type IIcollagen in vivo.
 5. The method of claim 4, further comprising the stepof contacting the serum sample with a second antibody which binds to abone resorption marker selected from among amino-terminal telopeptidesof type I collagen, carboxy-terminal telopeptides of type I collagen,and free lysly pyridinoline cross-links, and correlating any detectedbinding of the second antibody in the sample to bone resorption in vivo.6. The method of claim 4, further comprising the step of contacting theserum sample with a second antibody which binds to a type III collagenresorption marker selected from among amino-terminal telopeptides oftype III collagen and carboxy-terminal telopeptides of type IIIcollagen, and correlating any detected binding of the second antibody inthe sample to type III collagen resorption in vivo.
 7. In a method ofanalyzing a body fluid sample for the presence of an analyte indicativeof a physiological condition, comprising the steps of contacting thebody fluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a urine sample with an antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding to resorption of both unminemlized andmineralized type II collagen in vivo.
 8. The method of claim 7, furthercomprising the steps of determining the creatinine content of the urinesample and correlating the ratio of the detected binding of the antibodyto the creatinine content in order to provide a urinary index of totaltype II collagen resorption independent of urine volume.
 9. The methodof claim 7, further comprising the step of contacting the urine samplewith a second antibody which binds to a bone resorption marker selectedfrom among amino-terminal telopeptides of type I collagen,carboxy-terninal telopeptides of type I collagen, and free lysylpyridinoline cross-links, and correlating any detected binding of thesecond antibody in the sample to bone resorption in vivo.
 10. The methodof claim 7, further comprising the step of contacting the serum samplewith a second antibody which binds to a type III collagen resorptionmarker selected from among amino-terminal telopeptides of type IIIcollagen and carboxy-terminal telopeptides of type III collagen, andcorrelating any detected binding of the second antibody in the sample totype III collagen resorption in vivo.
 11. In a method of analyzing abody fluid sample for the presence of an analyte indicative of aphysiological condition, comprising the steps of contacting the bodyfluid sample with an antibody which binds to the analyte, detectingbinding of the antibody in the body fluid sample, and correlating anydetected binding to the physiological condition, the improvementcomprising contacting a serum sample with a first antibody which bindsto

and contacting the serum sample with a second antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating the total detected binding of the first and secondantibodies to resorption of both mineralized and unmineralized type IIcollagen in vivo.
 12. The method of claim 11, further comprising thestep of contacting the serum sample with a third antibody which binds toa bone resorption marker selected from among amino-terminal telopeptidesof type I collagen, carboxy-terminal telopeptides of type I collagen,and free lysyl pyridinoline cross-links, and correlating any detectedbinding of the third antibody in the sample to bone resorption in vivo.13. The method of claim 11, further comprising the step of contactingthe serum sample with a third antibody which binds to a type IIIcollagen resorption marker selected from among amino-terminaltelopeptides of type III collagen and carboxy-terminal telopeptides oftype III collagen, and correlating any detected binding of the thirdantibody in the sample to type III collagen resorption in vivo.
 14. In amethod of analyzing a body fluid sample for the presence of an analyteindicative of a physiological condition, comprising the steps ofcontacting the body fluid sample with an antibody which binds to theanalyte, detecting binding of the antibody in the body fluid sample, andcorrelating any detected binding to the physiological condition, theimprovement comprising contacting a serum sample with a first antibodywhich binds to

and contacting a urine sample with a second antibody which binds to

wherein K—K—K is hydroxylysyl pyridinoline or lysyl pyridinoline, andcorrelating any detected binding of the first antibody to resorption ofmineralized type II collagen in vivo, and any detected binding of thesecond antibody to resorption of both mineralized and unmineralized typeII collagen in vivo.
 15. The method of claim 14, further comprising thesteps of determining the creatinine content of the urine sample andcorrelating the ratio of the detected binding of the second antibody tothe creatinine content in order to provide a urinary index of total typeII collagen resorption independent of urine volume.
 16. The method ofclaim 14, further comprising the step of contacting at least one of theserum and urine samples with a third antibody which binds to a boneresorption marker selected from among amino-terminal telopeptides oftype I collagen, carboxy-terminal telopeptides of type I collagen, andfree lysyl pyridinoline cross-links, and correlating any detectedbinding of the third antibody in the sample to bone resorption in vivo.17. The method of claim 14, further comprising the step of contacting atleast one of the serum and urine samples with a third antibody whichbinds to a type III collagen resorption marker selected from amongamino-terminal telopeptides of type III collagen and carboxy-terminaltelopeptides of type III collagen, and correlating any detected bindingof the third antibody in the sample to type III collagen resorption invivo.
 18. Synthetic peptide DEKAGGA (SEQ ID NO:21).
 19. Syntheticpeptide GGFDEKAGGAQLG (SEQ ID NO:27).